Interferons differentially regulate histamine and TNF-alpha in rat intestinal mucosal mast cells.

Mast cell (MC) heterogeneity has been well characterized in the rat where it has been shown that connective tissue MC, often represented by peritoneal MC (PMC), and the intestinal mucosal MC (IMMC) exhibit many differences in mediator content and responsiveness to secretagogues and anti-allergic drugs. Pretreatment (20 hr) of PMC with interferon (IFN)-alpha/beta or IFN-gamma, significantly reduced antigen-stimulated histamine release. By contrast, for IMMC, the same IFN treatment did not modify antigen-stimulated histamine secretion. Although IFN treatment differentially modulates histamine secretion from PMC and IMMC, pretreatment of both MC types with IFN-alpha/beta or IFN-gamma inhibited their tumour-necrosis factor-alpha (TNF-alpha)-dependent cytotoxicity. In 20 hr of culture, IMMC spontaneously released 98 pg/10(6) MC of TNF-alpha, whereas PMC released about threefold more TNF-alpha, 282 pg/10(6) MC. In addition, direct assessment of stored TNF-alpha established that IMMC store less TNF-alpha (68 pg/10(6) MC) than PMC (404 pg/10(6) MC). In summary, TNF-alpha content of PMC and IMMC was different, but IFN inhibited TNF-alpha-dependent cytotoxicity by both MC types. By contrast, treatment with IFN-alpha/beta or IFN-gamma inhibits antigen-induced histamine secretion by PMC, but does not modify antigen-induced histamine secretion by IMMC. Thus, IFN differentially regulate the secretion of histamine and TNF-alpha in PMC and IMMC.     

Inhibition of tumour necrosis factor-alpha (TNF-alpha) release from mast cells by the anti-inflammatory drugs, sodium cromoglycate and nedocromil sodium.

TNF-alpha is a cytokine thought to be involved in the pathogenesis of asthma and in several other inflammatory conditions. Given recent evidence that mast cells (MC) are an important source of TNF-alpha, we investigated the effects of two anti-inflammatory drugs, nedocromil sodium (NED) and sodium cromoglycate (SCG), on rat MC-derived TNF-alpha. We established that at least 2 h pretreatment with NED or SCG followed by washing was required to inhibit TNF-alpha-dependent cytotoxicity by rat peritoneal MC (PMC). A maximum inhibition of TNF-alpha occurred after 6 h treatment. The inhibitory effect of NED and SCG (10(-5)-10(-3)M) was concentration-dependent (20-37% for NED and 16-37% for SCG). The time-course analysis and the use of cycloheximide, an inhibitor of protein synthesis, provided strong evidence that new protein synthesis by the MC is required for this inhibitory effect. Furthermore, 24 h treatment with 1 mM NED inhibited the levels of mRNA for TNF-alpha by 59-83%. In addition to the effect on TNF-alpha-dependent cytotoxicity by MC, 20 min pretreatment with 10(-4) M NED and SCG inhibited antigen-stimulated TNF-alpha release (6h) by 42% and 48%, respectively. Interestingly, the functionally distinct intestinal mucosal MC (IMMC) is unresponsive to these drugs with regard to histamine secretion. However, as with PMC, 2h pretreatment with NED or SCG inhibited TNF-alpha-dependent cytotoxicity by IMMC. These effects may be important in the action of these drugs in vivo in the late phase reaction in asthma or other inflammatory conditions.     

Mast cell response to formaldehyde. 1. Modulation of mediator release.

To examine the effects of the atmospheric pollutant formaldehyde on functionally distinct mast cells, peritoneal mast cells (PMC), intestinal mucosal mast cells (IMMC) and mouse bone-marrow-derived mast cells (BMMC) were incubated with various concentrations of formaldehyde. Pretreatment for 30 min with up to 100 micrograms/ml formaldehyde was not cytotoxic to mast cells. Formaldehyde (1-10 micrograms/ml) alone induced low levels of histamine release (< 10%) from IMMC and BMMC. Antigen-induced histamine release was significantly increased in both PMC pretreated with low concentrations of formaldehyde (5-20 micrograms/ml) and BMMC pretreated with 10 micrograms/ml formaldehyde but decreased in PMC pretreated with a higher concentration (100 micrograms/ml) of formaldehyde. By contrast, antigen-induced histamine release was decreased in IMMC pretreated with formaldehyde in a dose-dependent manner. Histamine release stimulated with A23187 was also increased in PMC pretreated with a low concentration (10 micrograms/ml) of formaldehyde but decreased in those pretreated with a higher concentration (100 micrograms/ml) of formaldehyde. Pretreatment with 10 micrograms/ml formaldehyde significantly enhanced beta-hexosaminidase release from PMC stimulated with antigen or A23187. Compared to sham-treated PMC, PMC pretreated with formaldehyde expressed a markedly depressed natural cytotoxicity for the tumor target WEHI-164 (an assay of tumor necrosis factor alpha activity). These results suggest that formaldehyde modifies various mast cell functions through alterations in cellular metabolism. Such effects may be important in respiratory and other diseases associated with formaldehyde exposure.     

A kinetic analysis of the expression of mast cell protease mRNA in the intestines of Nippostrongylus brasiliensis -infected rats

To study the kinetics and the phenotype of the mast cells (MC) arising during infection with the nematode Nippostrongylus brasiliensis, monospecific cDNA probes for nine different MC proteases were used in a Northern blot analysis of RNA from the small intestine of infected rats. The expression was analyzed at four individual time points during infection, day 0 (before infection), and days 7, 12 and 16 post infection. A dramatic increase in mRNA for rat mast cell protease (RMCP)-2, the major mucosal MC protease in the rat, was observed, beginning around day 7 after infection and peaking around day 12. At day 16 the expression was already beginning to decline. An almost identical pattern of mRNA expression was detected for the RMCP-8 subfamily of rat MC proteases (RMCP-8, −9 and −10) and for two additional rat serine proteases, the chymases RMCP-3 and −4. No simultaneous increase in the proteases known to be expressed preferentially by mature connective tissue MC (RMCP-1, −6 and −7) was observed. This is consistent with our finding that the expansion of MC in the intestines of parasite-infected animals was limited, almost exclusively, to the mucosal MC population. However, a minor increase in RMCP-5 and MC carboxypeptidase A (CPA) mRNA was detected at day 12 after infection, suggesting a derivation of mucosal MC from an expanding RMCP-5- and CPA-positive population of MC precursors.     

IgE receptors from rat intestinal mucosal and peritoneal mast cells show mast cell subtype-specific differences

High- (alpha chain) and low-affinity IgE receptors from purified populations of rat intestinal mucosal (IMMC) and peritoneal mast cells (PMC) were characterized by SDS-PAGE. Receptor expression and molecular weight were compared. IMMC yielded 59-kilodalton (kDa) alpha chains of the high-affinity receptors and two forms (58, 50 kDa) of low-affinity receptors, whereas PMC possessed only 51-kDa alpha chains and 56-kDa low-affinity receptors. These differences extend the evidence for functional diversity between mast cell subtypes.     

Isolation and characterization of lung mast cells from rats with bleomycin-induced pulmonary fibrosis

To study the nature and extent of mast cell heterogeneity within a single species, we have developed methodologies to isolate rat lung mast cells (LMC) and have compared these to peritoneal mast cells (PMC) and intestinal mucosal mast cells (IMMC). In normal and athymic nude (rnu/rnu) rats, a single intratracheal administration of bleomycin (5 U/kg) leads to pulmonary fibrosis accompanied by parenchymal hyperplasia of mast cells that are histochemically like PMC rather than IMMC. Using collagenase digestion of fibrotic rat lungs (30-80 days after bleomycin treatment), we recovered an average of 58.1 x 10(6) viable cells per rat, containing 2.5% mast cells. Control experiments in which PMC were subjected to the isolation procedure used for LMC showed that there was no qualitative effect on PMC, but that a reduction of 26-60% in responsiveness to secretagogues occurred. Isolated LMC secreted histamine in response to 48/80, A23187, substance P, VIP and somatostatin and bradykinin, but at lower levels than PMC. The anti-allergic compound theophylline, which does not inhibit antigen-induced histamine secretion by IMMC, was effective against both LMC and PMC. Taken together, the thymus independence of pulmonary mast cell hyperplasia, the histochemical characteristics and the responsiveness to secretagogues and anti-allergic compounds indicate that the majority of dispersed LMC are similar to PMC rather than to IMMC. Whether LMC should be considered analogous to PMC or, because of their size, histamine content and responsiveness to many secretagogues, intermediate between PMC and IMMC, remains to be determined through additional studies.     

Isolation and characterization of IgE receptors from rat intestinal mucosal mast cells

High-(Fc epsilon RI) and low-(Fc epsilon RII) affinity IgE receptors were isolated from surface radioiodinated, Nonidet-P40-solubilized rat intestinal mucosal mast cells (IMMC) and compared with those on rat peritoneal mast cells (PMC) and rat basophilic leukemia (RBL) cells. Fc epsilon RII were isolated by affinity chromatography using IgE-Sepharose or by anti-Fc epsilon RII antisera and protein A-Sepharose. The surface-exposed, IgE-binding alpha subunits of Fc epsilon RI [Fc epsilon RI alpha] were isolated by affinity chromatography using IgE and anti-IgE-Sepharose. Fc epsilon RI alpha on IMMC had an apparent molecular mass of 59 kDa, somewhat larger than that of PMC (51 kDa), RBL-2H3 cells (51 kDa) or RBL-CA10.7 cells (46 kDa). Brief (45 s) incubation of IMMC or PMC in glycine-HCl, pH 3, prior to iodination removed much of the surface-bound IgE. This permitted more thorough labeling of the receptors, but had no affect on the estimate of receptor size. Surprisingly and in contrast to acid-treated PMC, upon anti-IgE-Sepharose isolation acid-treated IMMC yielded an intensely radioactive Fc epsilon RI alpha band in the absence of added IgE. Such a finding suggests that IMMC, more so than PMC, may have an intracellular store of IgE, as has been suggested by many others. IMMC also differed from PMC in the number of forms of Fc epsilon RII isolated; 50-kDa and 58-kDa forms of Fc epsilon RII were obtained from IMMC, whereas PMC yielded most often a single 56-kDa Fc epsilon RII band. These results were mimicked by the two RBL cell sublines: RBL-2H3 cells yielded two Fc epsilon RII (46 kDa and 55 kDa), but only one form of Fc epsilon RII (54-kDa) was obtained from RBL-CA10.7 cells. Thus, the two subtypes of rat mast cells, which have previously been shown to differ in mediator profile and responsiveness to secretagogues and antiallergic drugs, are also distinguished by differences in IgER profile.     

IgE-mediated release of rat mast cell protease II, beta-hexosaminidase and leukotriene C4 from cultured bone marrow-derived rat mast cells.

Functional characteristics of cultured bone marrow-derived rat mast cells (BMMC) were studied. BMMC were shown to release in a time- and dose-dependent fashion the mucosal mast cell (MMC)-specific enzyme, rat mast cell protease II (RMCPII), following IgE-mediated activation in vitro. RMCPII release was temporally associated with that of the mast cell granule-derived enzyme, beta-hexosaminidase (beta-hex). Release of the pre-formed granule constituents, RMCPII and beta-hex, was associated with the generation of the membrane-derived lipid mediator, leukotriene C4 (LTC4) and, in older cultures, substantial amounts were generated (25.2 ng/10(6) BMMC). Absolute amounts of RMCPII, beta-hex and LTC4 released were dependent upon the age of the BMMC. These results extend our previous observations on the staining properties and protease content of rat BMMC and provide evidence that these cells are functionally, as well as histochemically, analogous to the MMC subset, which is so prominent during intestinal nematode infections in rats.     

Mast cell heterogeneity: two-dimensional gel electrophoretic analyses of rat peritoneal and intestinal mucosal mast cells

Peritoneal mast cells (PMC) and intestinal mucosal mast cells (IMMC) were purified from rats infected with the nematode Nippostrongylus brasiliensis. Overall protein constituents of both mast cell subtypes were analyzed by two-dimensional gel electrophoresis using either nonequilibrium pH gradient electrophoresis (NEPHGE) or isoelectric focusing (IEF) in the first dimension and SDS-PAGE (10%) in the second dimension followed by silver staining. PMC had seven dominant basic proteins (PB2-8; pI 9-9.5) with estimated molecular masses of 26 to 37 kDa, as well as 80 to 90 neutral or acidic proteins, most of which had pI 6 to 7.5 and estimated molecular masses of 20 to 100 kDa. All the basic proteins were granule-associated. Three basic proteins, PB6 (29 kDa), PB7 (28 kDa) and PB8 (RMCP I, 26 kDa), bound [3H]diisopropyl fluorophosphate (DFP), suggesting that they are serine proteases. However, only PB8 was reactive with antibodies to RMCP I. Another basic component (less than 14 kDa), perhaps a degradation product of PB6, PB7 or PB8, also bound [3H]DFP. By comparison, IMMC possessed nine basic proteins (IB1-9) and, in general, they were more acidic (pI about 8.5-9) than those of PMC. Four major basic proteins (IB6-9) were all 24 kDa but were slightly different in isoelectric points. These and another 46-kDa basic component (IB2) were reactive with antibodies to RMCP II and bound [3H]DFP. There were no other DFP-binding proteins in IMMC. In spite of remarkable differences between basic granule-associated proteins in PMC and basic proteins in IMMC, spots in the neutral-acidic range were for the most part similar in the two mast cell subsets, although quantitative differences were evident for some spots. Thus, rat mast cell populations from the peritoneal cavity and intestinal mucosa exhibit marked heterogeneity in their protein constituents with basic pI, including in their granule-associated proteins with serine protease activity.     

Mast cell heterogeneity: protein composition, biosynthesis and mRNA characterization

Proteins of rat peritoneal mast cells (PMC), rat intestinal mast cells (IMMC) and human skin mast cells (SMC) were compared by two-dimensional electrophoresis. PMC and IMMC had many similarities in distribution of their neutral/acidic proteins but marked differences in the more abundant, granule-associated basic proteins. SMC proteins showed a unique distribution. Distributions of the products of in vitro translation of PMC and IMMC RNA were different from those of proteins isolated directly from these cells which, together with results of pulse-chase labelling experiments, shows evidence for processing of certain mast cell proteins including rat mast cell proteases I and II.     

Functional characterization of mast cells generated in vitro from the mesenteric lymph node of rats infected with Nippostrongylus brasiliensis.

We have examined the biochemical and functional characteristics of mast cells grown in tissue culture from the mesenteric lymph node (MLN) of rats infected with the nematode Nippostrongylus brasiliensis and compared them with mast cells isolated from the small intestinal mucosa and peritoneal cavity of infected animals. Cultured mast cells (MC) and isolated intestinal mucosal mast cells (MMC) had a similar histamine content, and both contained type II protease (RMCP II) which was absent from peritoneal mast cells (PMC). PMC, MMC and cultured MC each responded to immunologically induced histamine secretion, but MMC and cultured MC were hyporesponsive to calcium ionophores and unresponsive to widely used PMC secretagogues including compound 48/80 and bee venom peptide 401. MMC and cultured MC also differed from PMC in their lack of responsiveness to the anti-allergic agent disodium cromoglycate. Thus, MC cultured from the MLN are distinct from PMC but have a biochemical and functional phenotype similar to that of intestinal MMC.     

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs.     

The action of tumor necrosis factor-alpha on rat mast cells.

Taking into account that cytokine tumor necrosis factor-alpha (TNF-alpha) and mast cells (MC) both are involved in inflammation, it seems of great importance to recognize their relationships. Therefore, we have studied whether recombinant human TNF-alpha (rHuTNF-alpha) can cause histamine secretion from rat peritoneal MC. We have also examined the effect of this cytokine on MC reactivity. We have established that TNF-alpha stimulates rat MC to histamine release in a concentration-dependent manner. TNF-alpha-induced histamine secretion was evoked by concentrations > 10-16 M and reached the maximum rate at a concentration of 10-10 M (histamine release 17.1% +/- 1.9%, mean +/- SEM). We have also noticed that pretreatment of MC with TNF-alpha (in a concentration of 10-16 M) significantly inhibited concanavalin A (ConA)-stimulated release of histamine, with the percent release decreasing to 51% of the control value. Treatment of mast cells with TNF-alpha resulted in a decrease of compound 48/80-dependent histamine release as well (the percent released histamine fell to 85% of the control value). This altered MC responsiveness was reversible. After 120 min of resting time, the MC reactivity came back to the initial values. We have concluded that TNF-alpha appears to be a direct stimulus for MC to release histamine, and it may regulate MC secretory function.     

Stem cell factor and IgE-stimulated murine mast cells produce chemokines (CCL2, CCL17, CCL22) and express chemokine receptors

OBJECTIVE AND DESIGN: In the present study we investigated the effect of SCF and/or IgE on histamine, TNF-alpha and chemokines released from bone marrow-derived mast cells (BMMC) as well as chemokine receptor expression. MATERIAL AND METHODS: BMMC were derived from femoral bone marrow of CBA/J mice. The purity of BMMC was >98% after 3 weeks. BMMC (2.5 x 10(6) cells/well) were incubated in the presence or absence of either SCF, IgE plus DNP or a combination of SCF and IgE for 6 and 18 h. Cell-free supernatants were recovered to measure CC chemokines, TNF-alpha and histamine release utilizing ELISA assays. CC chemokine family receptors were detected by RT-PCR analysis, and confirmed using functional chemotactic assays. RESULTS: Histamine levels were comparable between SCF and IgE stimulated cells, whereas TNF-alpha production was significantly greater after IgE compared to SCF stimulation. SCF and/or IgE-stimulated BMMC released CC chemokines, CCL22 (MDC), CCL17 (TARC) and CCL2 (MCP-1). Increased mRNA expression of CCR1, CCR2, CCR3, and CCR5 was detected in SCF and IgE-stimulated BMMCs. Functional chemotactic assays confirmed the expression data. CONCLUSION: SCF and IgE can up-regulate the expression of chemokines and chemokine receptors on mast cells. Thus, SCF may play a significant role in their activation and inflammation during allergic responses.     

The Src kinase Lyn is a negative regulator of mast cell proliferation

Previous investigators have reported that deletion of the protein tyrosine kinase Lyn alters mast cell (MC) signaling responses but does not affect or reduces the cytokine-mediated proliferation of mouse bone marrow-derived MC (BMMC) precursors and of mature MC. We observed that Lyn-deficient mice have more peritoneal MC than wild-type (WT) mice. Studies to explore this unexpected result showed that Lyn(-/-) BM cells expand faster than WT cells in response to interleukin (IL)-3 and stem-cell factor over the 4-5 weeks required to produce a >95% pure population of granular, receptor with high affinity for immunoglobulin E-positive BMMC. Furthermore, differentiated Lyn(-/-) BMMC continue to proliferate more rapidly than WT BMMC and undergo less apoptosis in response to cytokine withdrawal. Additionally, Lyn(-/-) BMMC support greater IL-3-mediated phosphorylation of the prosurvival kinase, Akt, and the proliferative kinase, extracellular-regulated kinase 1/2. These results identify Lyn as a negative regulator of murine MC survival and proliferation.     

肥大细胞对眼眶成纤维细胞生长的影响

目的:探讨肥大细胞对眼眶成纤维细胞生长的影响。方法:将分离纯化的小鼠骨髓源性肥大细胞(BMMC)与眼眶成纤维细胞(OFb)共育,共育分为两组:两种细胞接触共育组和非接触共育组,眼眶成纤维细胞单独培养为对照组;应用光镜、电镜及细胞生长计数等方法研究肥大细胞对眼眶成纤维细胞生长的影响。结果:与BMMC接触共育的OFb增殖活跃,呈复层生长,单位面积内细胞计数明显增加;接触共育组中OFb的增殖数分别与非接触共育组和对照组比较,差异均有统计学意义(P<0.01);而非接触共育组的OFb增殖数和对照组比较,差异没有统计学意义(P>0.05);电镜见接触共育组OFb中粗面内质网(RER)增生扩张,BMMC与OFb紧密接触,部分BMMC有脱颗粒现象。结论:肥大细胞对眼眶成纤维细胞的生存和增殖有显著的促进作用,并使其功能活跃,这种作用主要是通过两种细胞间的紧密接触而实现的。     

Stem cell factor-dependent survival, proliferation and enhanced releasability of purified mature mast cells isolated from human intestinal tissue

BACKGROUND: Recently we reported about a stem cell factor (SCF)-dependent culture system for human mast cells (MC), isolated from intestinal mucosa. Here we present a method to obtain highly purified human intestinal MC. METHODS: MC were isolated from surgery specimens and purified by positive selection using the magnetic-activated cell sorting (MACStrade mark) system and subsequent culture of the MC in medium supplemented with SCF. RESULTS: In the presence of SCF, purified MC (50-85% purity after MACS) maintained in culture for up to 3 months. MC purity increased during culture and reached nearly 100%. During the first week of culture, MC numbers decreased, but after that time they started to proliferate. Cultured MC did not change their histamine content, phenotype or morphology. They were even more responsive towards IgE-dependent stimulation, which caused the release of high amounts of histamine, leukotrienes and cytokines such as TNF-alpha and IL-5. CONCLUSION: We show that mature human intestinal MC can be purified, maintained in culture, and triggered for proliferation in the presence of SCF. After culture, they are viable, release high amounts of mediators and cytokines upon stimulation, and thus are a valuable tool for further experiments on human mucosal MC.     

Nonspecific B and T cell-stimulatory activity mediated by mast cells is associated with exosomes

Bone marrow-derived mouse mast cells (BMMC) and mast cell lines P815 and MC9 have recently been shown to induce antigen-independent B and T lymphocyte activation. It has been demonstrated that a physical contact between mast cells and B and T lymphocytes is not necessary since mast cell supernatants contain full activity. Electron microscopy studies revealed the presence in mast cell supernatants of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induce B and T cell blast formation, proliferation as well as IL-2 and IFN-gamma production. In contrast to P815 and MC9 mast cell lines, a pretreatment with IL-4 is required for BMMC to produce active exosomes. Structurally, these exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1 and ICAM-1. Here we provide for the first time the evidence that mast cells use exosomes as sophisticated messengers to communicate with cells of the immune system.